Nutraceuticals are foods that have specific medicinal as well as nutritional benefits. One nutraceutical, kappa-casein (.kappa.-casein) macropeptide comprises 15-20% of the protein in whey, making its supply plentiful. Widely differing extents of glycosylation of .kappa.-casein macropeptide (CMP) exist in whey and whey products, ranging from fully-glycosylated CMP (called .kappa.-casein glycomacropeptide, or GMP) to non-glycosylated CMP. For purposes of the present invention, CMP includes all forms of the .kappa.-casein macropeptide from the fully-glycosylated .kappa.-casein glycomacropeptide to the non-glycosylated .kappa.-casein glycomacropeptide. As discussed by Shammet et al. (1992), total CMP, which includes all degrees of glycosylation, is measured as non-protein nitrogen (NPN-2) soluble in 2% trichloroacetic acid (TCA). GMP is measured as non-protein nitrogen soluble in 12% TCA (NPN-12). The difference between NPN-2 and NPN-12 is non-glycosylated CMP.
CMP is the moiety cleaved from .kappa.-casein at the Phe.sup.105 -Met.sup.106 position by chymosin during cheese making (Dalgleish, 1982; Fox, 1989). CMP occurs at a concentration of 1.2 to 1.5 g/L in sweet whey (Cheddar cheese whey), comprising 15 to 20% of the total protein (Marshall, 1991). Kawaski et al. reviewed known biological functions of GMP as consolidated from several nutritional and biological research reports (U.S. Pat. No. 5,278,288 to Kawaski and Dosako; Kawaski et al., 1992), and Tanimoto et al. (1992) suggested the utilization of GMP in dietetic foods and pharmaceuticals. The advantage of CMP as a substrate for hydrolysis is that it does not contain the amino acids tyrosine, phenylalanine, and tryptophan. These are hydrophobic aromatic amino acids which make peptides bitter. Alternatively, Marshall (1991) suggested that CMP can be used as a protein source for the treatment of phenylketonuria, a hereditary disorder in which aromatic amino acids cannot be metabolized.
On a laboratory scale, CMP has been purified from .kappa.-casein in bovine whey using TCA precipitation (Shammet et al., 1992). GMP has been purified from bovine whey by alcohol precipitation after heat coagulation of whey protein (Saito et al., 1991). These methods are unlikely to be economical for large-scale manufacturing.
On a large scale, several methods have been developed using ultrafiltration (Kawakami et al., 1992; Kawaski et al., 1993; Tanimoto et al., 1990) and ion exchange (U.S. Pat. No. 5,278,288 to Kawaski and Dosako; U.S. Pat. No. 5,290,107 to Kawaski et al.) to purify GMP from whey or whey protein concentrate. However, the recovery of GMP using these methods is uneconomically low, at most 18% (U.S. Pat. No. 5,278,288 to Kawaski and Dosako; U.S. Pat. No. 5,290,107 to Kawaski et al.). The basis of ultrafiltration purification is that the apparent molecular weight of GMP is 10 to 30 kDa at pH 3.5 and 20 to 50 kDa at pH 7.0. At pH 3.5, GMP permeates 20 to 50 kDa molecular weight cutoff (MWCO) ultrafiltration systems while proteins are retained by the membrane. Then at pH 6.5, GMP is retained by the same MWCO membranes while small molecular weight contaminants such as peptides pass through the filter.
U.S. Pat. Nos. 5,278,288 and 5,280,107 to Kawasaki et al. are specifically directed to GMPs. In the '288 patent, the GMP flows through a concentrating and desalting step unadsorbed. The '107 patent requires a pH value of about 4 or lower. Neither patent mentions the hydrolysis of GMP to form a solution depleted in aromatic amino acids, the presence of which causes bitterness. Neither patent mentions the use of two ion exchangers in series for the adsorption of GMP.
GMP contains substantial amounts of covalently bound sialic acids, which strongly determine the binding behavior in ion exchange separations. For example, the '107 patent to Kawasaki et al. discloses that because the sialic acids present in GMP have a pKa value of 2.7, these moieties have a net negative charge at pH as low as 3 to 4. In non-glycosylated CMP, the potentially negatively-charged amino acid side chains (aspartic and glutamic acid) have a pKa of 3 to 5, and would have a substantial net negative charge only at pH 5 and higher. The presence of sialic acids allowed Kawasaki et al. to separate GMP from proteins which lack sialic acids, by adsorbing the GMP to an anion exchanger at pH 4 or lower, because the other proteins are neutrally or positively charged at this pH. However, the method of Kawasaki et al. would not be suitable for recovering all of the CMP from whey because non-glycosylated CMP would not bind strongly to an anion exchanger until it had a substantial net negative charge, which would occur only at pH 5 or higher. Because some of the uses for CMP are based on the absence of aromatic amino acids, a characteristic of all CMP, methods specific for the recovery of only GMP would not be suitable for the full recovery of all CMP.
U.S. Pat. No. 5,075,424 to Tanimoto et al., U.S. Pat. No. 5,061,622 to Dosako et al., U.S. Pat. No. 5,216,129 to Berrocal et al. also describe processes for producing GMPs.